Hedgehog (Shh or Hh), WNT, FGF and BMP signaling pathways network together during embryogenesis, tissue regeneration, and carcinogenesis. Aberrant activation of Hh signaling pathways leads to pathological consequences in a variety of human tumors, such as gastric cancer and pancreatic cancer. Hedgehogs are secreted glycoproteins that initiate Hh signal transduction by binding to a transmembrane protein complex comprising PATCHED1 (ptch1) and SMOOTHENED (smo) and eliciting a cascade of cytoplasmic signal transduction events, including the inhibition of a protein kinase A that leads to the transcription of the GLI zinc-finger transcription factors. The GLI family of zinc-finger transcription factors then translate the extra-cellular Hh-stimulus into defined transcriptional programs in a context-dependent and cell-type specific manner (Ruiz I Altaba et al., 2002, Nat. Rev. Cancer 2:361-72).
Several proteins, including GLI proteins, are involved in mediating Hh signaling (Katoh and Katoh, 2005, Cancer Biol. Ther. 4:1050-4). Vertebrates have at least three distinct GLI proteins, GLI (also referred to as GLI1), GLI2, and GLI3. These proteins are members of the GLI family of zinc finger transcription factors and share a highly conserved C2—H2 zinc finger domain (having five zinc finger DNA-binding motifs) with Drosophila Cubitus interruptus (Ci) and the Caenorhabditis elegans sex-determining gene tra-1 (Hui et al., 1994, Dev. Biol. 162:402-13). In Drosophila Ci is required for activation of hedgehog targets and also functions as a repressor of hedgehog expression.
During embryonic development of vertebrates all genes whose expression is partially overlapping are transcriptionally activated in response to Hh-signaling and are able to mediate most of the effects caused by activation of the pathway. GLI1, GLI2 and GLI3 may each carry out a specific function during vertebrate development (Kinzler et al., 1984, Nature 332:371-374; Ruppert et al., 1988, Mol. Cell. Biol. 8:3104-3113; Walterhouse et al., 1993, Dev. Dyn. 196:91-102; Hui et al., 1994, Dev. Biol. 162:402-413). For example, Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum (Mo et al., 1997, Development 124:113-23).
Gli3 represents an important control gene for development and differentiation of several body structures. For example, a reduction in gene dosage leads to severe perturbation, especially limb morphogenesis (Vortkamp et al., 1995 DNA Cell Biol. 14:629-34). Further, mutations in GLI3 have been identified in several human malformation syndromes, such as Greig cephalopolydactyly syndrome (GCPS) affecting limb and craniofacial development and the autosomal dominant form of Pallister-Hall syndrome. (Vortkamp et al., 1991, Nature 352:539-40; Bose et al., 2002, Hum. Mol. Genet. 11:1129-35). In the mouse, GLI3 mutations have been implicated in the mouse mutant extra toes.
GLI proteins function as transcriptional activators by binding via their zinc finger domains to a DNA binding site within a promoter and/or enhancer sequence of target genes. Recently, DNA binding sequences bound by GLI proteins were identified. For example, the DNA sequence bound by GLI3 zinc fingers consists of 16 nucleotides and shows a high degree of similarity to sequences bound by the GLI and tra-1 proteins (Vortkamp et al., 1995, DNA Cell. Biol. 14:629-34). This binding site included the 9-base-pair sequence 5′-GACCACCCA-3′ previously identified for GLI protein binding (Kinzler and Vogelstein, 1990, Mol. Cell. Biol. 10:634-42). It is believed that GLI, GLI2, and GLI3 bind identical or similar DNA sequences (Yoon et al., 1998, J. Biol. Chem. 273:3496-3501). Recently, the 9 bp consensus sequence 5′-GACCACCCA-3′ as a binding site for GLI1, GLI2, and GLI3 proteins was confirmed by Hallikas et al. (Hallikas et al, 2006, Cell 124:47-59).
Activation of Hh/GLI target genes by GLI2 and GLI3 proteins may require the transcriptional co-activator Creb Bing Protein (CBP), which binds to a CBP binding domain (see FIG. 1; Dai et al., 1999, J. Biol. Chem. 274:8143-52; Kasper, Regl, Frischauf and Aberger, 2006, Eur. J. Cancer)
GLI1 expression has been reported to be under control of GLI2 and GLI3 proteins Dai et al., 1999, J. Biol. Chem. 274:8143-52). While the whole GLI1 protein is a transcriptional activator, GLI2 and GLI3 contain both activator and repressor domains (see FIG. 1). GLI2 and GLI3 can be processed for different functions through the action of protein kinase A (PKA). Full-length GLI2 acts as a weak transcriptional activator. Truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N-terminus converts GLI2 into a strong activator. N-terminally truncated GLI2, unlike the full length protein, activates, for example a Sonic hedgehog (Shh) gene, HNF3beta, in transgenic mouse embryos. This suggested that unmasking the activation domain of GLI2 is one of the key mechanisms of the Shh signaling pathway. A similar regulatory mechanism involving the N-terminal region was also described for GLI3, but not for GLI. (Sasaki et al., 1999, Development 126:3915-24, incorporated by reference in its entirety).
A comparison of the murine and human GLI3 cDNA revealed an overall homology of 85% between the deduced amino acid sequences and an even higher conservation (<95%) in several domains, including the zinc fingers (Thien et al., 1996, Biochim. Biophys. Acta 1307:267-9).
Recently, a transcription activation domain for GLI was identified at the carboxy-terminus terminus which comprised amino acid residues 1020 to 1091. This domain includes an 18 amino acidic α-helix (amino acids 1037 to 1054) containing six aspartate or glutamate residues (Yoon et al., 1998, J. Biol. Chem. 273:3496-3501). This α-helical region is highly similar to the herpes simplex viral protein 16 (VP16) transcription activation domain, and includes the conserved motif FXXΦΦ (F=phenylalanine; X=any residue; Φ=any hydrophobic residue), described as a general recognition element of acidic activation domains for TAFII31. In addition, conservation of the three amino acid residues (Asp472, Phe479, and Leu483 in VP16, and Asp1040, Phe1048, and Leu1052 in GLI) that are believed to make direct contacts with the TBP-Associated Factor (TAF), TAFII31, was found (Yoon et al., 1998, J. Biol. Chem. 273:3496-3501; Goodrich et al., 1993, Cell 75:519-530; Klemm et al., 1995, Proc. Natl. Acad. Sci. USA 92:5788-5792; Uesugi et al., 1997, Science 277:1310-1313).
A similar domain is present in the other GLI family proteins. For example, putative TAFII31 binding domains were described and include NH2-INKDNLRKDLFTVSIKA-COOH (Drosophila Ci, amino acid residues 1044-1060), NH2-DMADFEFEQMFTDALGI-COOH (VP16, amino acid residues 469-485), NH2-DSLDLDNTQLDFVAILDE-COOH (human GLI, amino acid residues 1037-1054), NH2-DSLDLDNTQLDFVAILDE-COOH (mouse GLI, amino acid residues 1040-1057), NH2-DSHDLEGVQIDFDAIIDD-COOH (human GLI3, amino acid residues 1495-1512), and NH2-DSQLLEPPQIDFDAIMDD-COOH (mouse GLI2, amino acid residues 1509-1526). (Yoon et al., 1998, J Biol. Chem. 273:3496-3501). In addition, putative TAFII31 interaction domains of human GLI2, NH2-DSQLLEAPQIDFDAIMDD-COOH (amino acid residues 1501-1518) can be identified, e.g., from GenBank Accession No. AAY87165. Further, the putative TAFII31 interaction domain of mouse GLI3, NH2-ESHDLEGVQIDFDAIIDD-COOH (amino acid residues 1497-1514) can be identified from, e.g., GenBank Accession No. Q61602. The conserved amino acid residues for contacting TAFII31 are indicated in bold and underlined.
Though much is known about Hh-signaling in Drosophila and murine development, understanding of the molecular mechanisms and tumorigenic programs that are activated in response to Hh-signaling and GLI-activity in human cancer is still very limited. A common property of Hh-associated cancers is the elevated expression level of one or more GLI proteins. For example, recently, overexpression of GLI1 was found in a number of cancer types, including prostate, pancreas and small-cell lung cancer (Karhadkar et al., 2004, Nature 431:707-12). In addition, Gli was found to be amplified in childhood sarcomas and more than 50-fold in a malignant glioma (Roberts et al., 1989, Cancer Res. 49:5407-13; Kinzler et al., 1987, Science 236:70-3). Although Hedgehog (Hh) and its receptor Patched (Ptch) have also been found to be overexpressed in non small-cell lung cancer (NSCLC), no GLI1 expression was found in this type of cancer.
Overexpression of GLI2, for example, has been found in basal cell carcinoma (BCC) lesions compared to normal skin (Ikram et al., 2004, J. Invest Dermatol. 122:1503-9; Regl et al., 2002, Oncogene 21:5529-39; Hutchin et al., 2005, Genes Dev. 19:214-23). In addition, based on the processing of GLI2 and GLI3, GLI2 and/or GLI3 might be more important in activation of Hh signaling during tumorigenesis for many types of cancer, including lung cancer and prostate cancer.
Lung Cancer is the leading cause of cancer death in the United States and worldwide, with >170,000 newly diagnosed cases each year in the United States and nearly a million cases worldwide (Minna et al., 2002, Cancer Cell. 1(1):49-52). Despite aggressive approaches made in the therapy of lung cancer in the past decades, the 5-year survival rate for lung cancer remains under 15%. Lung cancers are divided into two groups: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC (75-80% of all cancers) consists of three major types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma (Minna et al., 2002, Cancer Cell. 1(1):49-52). Lung carcinomas and squamous cell carcinomas represent 60-70% of all lung cancers. Surgery, chemotherapy, and radiation have been used with generally unsatisfactory results in advanced disease. Improvement in the efficacy of lung cancer treatment is a major public health goal.
Prostate cancer may be the most common solid tumor in men (Nelson et al., 2003, N. Engl. J Med. 349:366-381). For example, 50% of all men over 50, and essentially all men over 70 suffer from some form of prostate hyperplasia. In the United States, prostate cancer is the most frequently diagnosed cancer, with over 250,000 new cases being diagnosed each year. 40,000 men die every year due to prostate cancer.
Thus, given the broad spectrum of frequent and lethal malignancies involving Hh/GLI-signaling, specific targeting of the Hh/GLI-signaling pathway may offer a highly effective therapeutic strategy for the treatment of a variety of lethal tumors (Pasca di Magliano and Hebrok, 2003, Nat. Rev. Cancer 3:903-911; Sanchez et al., 2005, Cancer Res. 65:2990-2; Kasper, Regl, Frischauf and Aberger, 2006, Eur. J Cancer). Treatment of human diseases resulting from ectopic Hh or GLI signaling pathway activation may require the use of pathway antagonists. Up to now, inhibition of ectopic activity has been achieved by treatment with signaling antagonists that block the pathway at different levels. For example, anti-Shh antibodies act extracellularly and the plant alkaloid, cyclopamine, acts at the level of Smo in the cell membrane (e.g., see U.S. Pat. Appl. No. 2005/0130922 A1; Taipale et al., 2000, Nature 406:1005-9; Sanchez and Ruiz I Altaba, 2005, Mech. Dev. 122(2):223-30; Athar et al., 2004, Cancer Res. 64(20):7545-52). Small molecule modulators of hedgehog signaling and binding to Smo were described, including a synthetic non-peptidyl small molecule, Hh-Ag (Frank-Kamenetsky et al., 2002, J. Biol. 1(2):10), HhAntag (Romer et al., 2004, Cancer Cell. 6(3):229-40), and Cur61414 (Williams et al., 2003, Proc. Natl. Acad. Sci. USA 100(8):4616-21). In addition, forskolin has been used to intracellularly activate protein kinase A (PKA), which is a cytoplasmic inhibitor of the GLI signaling pathway. However, these approaches have disadvantages. For example, administration of therapeutically effective amounts of anti Shh antibodies is difficult to achieve and may affect other normal pathway dependent cells in the patient. Cyclopamine, which is very expensive, may only be useful to treat disease that arise through activation of the Hh signaling pathway at the level of Smo or above. Further, because of the wide-spread activity of PKA, administration of forskolin may lead to numerous side effects. In contrast thereto, the use of small molecule compounds that inhibit GLI signaling holds great promise.
The inventors address a great and unfulfilled need in treating cancers wherein GLI proteins, and in particular GLI3, are overexpressed. The present invention provides small molecule compounds, pharmaceutical compositions, kits and methods useful for the detection and treatment of a number of cancers wherein GLI3 protein is overexpressed. Such cancers include lung cancer, NSCLC, breast cancer, colon cancer, mesothelioma, melanoma, sarcoma, prostate cancer, ovarian cancer, renal cancer, esophageal cancer, gastric cancer, hepatocellular cancer, nasopharyngeal cancer, pancreatic cancer, glioma, and others.